1. Field of the Invention
The present invention provides novel vaccines and diagnostic agents for the prevention, treatment and/or diagnosis of viral infection, especially papillomavirus infection and cervical cancers associated therewith. More specifically, the present invention provides an efficient method for incorporating immunotherapeutic proteins into capsomeres, which may then be used to elicit immune responses.
2. Description of the Prior Art
Papillomaviruses infect a wide variety of different species of animals including humans. Infection is typically characterized by the induction of benign epithelial and fibro-epithelial tumors, or warts at the site of infection. Each species of vertebrate is infected by a species-specific set of papillomavirus, itself comprising several different papillomavirus types. For example, more than sixty different human papillomavirus (HPV) genotypes have been isolated. Papillomaviruses are highly species-specific infective agents. For example, canine and rabbit papillomaviruses cannot induce papillomas in heterologous species such as humans. Neutralizing immunity to infection against one papillomavirus type generally does not confer immunity against another type, even when the types infect a homologous species.
In humans, papillomaviruses cause genital warts, a prevalent sexually-transmitted disease. HPV types 6 and 11 are most commonly associated with benign genital warts condylomata acuminata. Genital warts are very common, and subclinical or inapparent HPV infection is even more common than clinical infection. While most HPV-induced lesions are benign, lesions arising from certain papillomavirus types e.g., HPV-16 and HPV-18, can undergo malignant progression. Moreover, infection by one of the malignancy-associated papillomavirus types is considered to be a significant risk factor in the development of cervical cancer, the second most common cancer in women worldwide. Of the HPV genotypes involved in cervical cancer, HPV-16 is the most common, being found in about 50% of cervical cancers. The prevalence of HPV-18 ranges from approximately 8-31% depending on the geographical location, and in most areas worldwide, HPV-45 is the third most frequent, oncogenic HPV type (Bosch, F. X., et al., J Natl. Cancer Inst., 87:796-802 (1995).
In view of the significant health risks posed by papillomavirus infection generally, and human papillomavirus infection in particular, various groups have reported the development of recombinant papillomavirus antigens and their use as diagnostic agents and as prophylactic vaccines. In general, such research has been focused toward producing prophylactic vaccines containing the major capsid protein (L1) alone or in combination with the minor capsid protein (L2).
Yuan, et al., J Virology, 75:7848-53 (2001), describe the preparation of canine oral papillomavirus capsid L1 protein-glutathione-S-transferase fusion proteins and their expression in E. coli. This publication demonstrates the efficacy of non-VLP vaccines in the dog canine oral papillomavirus (COPV) model. COPV is the model previously used to validate VLP vaccines. Here, GST-L1 fusions were expressed in E. coli, and, although in capsomere form, had not been assembled into VLPs before use as a vaccine. The paper demonstrated that GST-L1 fusions retained their native conformations and further, completely protected dogs from viral infection with COPV. The authors conclude that VLPs are not necessary for efficacy of capsid protein L1 vaccines against papillomavirus, and that GST fusion proteins, which may be purified efficiently and economically in bacteria, are effective to protect dogs against COPV.
U.S. Pat. No. 6,165,471, issued Dec. 26, 2000 to Robert L. Garcea, et al., also discloses non-VLP vaccines composed of capsomeres of HPV L1 with carboxy-terminal deletions and mutations of specific cysteine residues which inhibit the formation of VLPs. These capsomeres were produced in bacterial expression systems, and they were efficacious in eliciting HPV neutralizing antibodies in rabbits. This reference is incorporated herein by reference in its entirety.
PCT/US01/18702 entitled “Stable (Fixed) Forms Of Viral Capsid Proteins, And Viral Capsid Protein Fusions, Preferably Papillomavirus L1 Proteins, And Uses Thereof,” describes papillomavirus capsid protein L1 or L2 proteins expressed as glutathione-S-transferase fusion proteins in bacteria. These fusions expressed in E. coli retain L1 native conformation and immunogenic activity as measured by assays with neutralizing antibodies. This reference is incorporated herein by reference in its entirety.
Prophylactic vaccines currently in clinical trials are based upon VLPs (virus like particles) assembled from HPV16 L1. See, Cain, J. M., et al., Science, 288:1753-55 (2000); Gissmann, L., et al., Intervirology, 44:167-75 (2001); Harro, et al., J. Natl Cancer Inst, 93:284-292 (2001); Schiller, J. T., et al., J Clin Virol, 19:67-74 (2000); and Schiller, J. T., et al., Expert Opin Biol Ther, 1:571-81. However, these types of vaccines are relatively expensive to produce in that they require eukaryotic expression systems or complex purification, and are less stable than capsomere preparations.
Additionally, VLP vaccines have the shortcoming that they may not provide cross protection against other papillomavirus serotypes, as neutralizing immune responses tend to be predominately type-specific. See Christensen, et al., J Gen Virol, 75(Pt 9):2271-76 (1994); Christensen, et al., Virology, 175:1-9 (1990); Roden, et al., J Virol, 70:5875-83 (1996); Roden, et al., J Virol, 70:3298-301 (1996); Rose, et al., J Gen Virol, 75 (Pt 9):2445-49 (1994); and White, et al., J Virol, 73:4882-9 (1999).
Papillomavirus capsid protein L2 has been shown to generate cross-neutralizing antisera. See Roden, “Minor capsid protein of human genital papillomaviruses contains subdominant, cross-neutralizing epitopes,” Virology, 270:254-7 (2000).
A further drawback to approaches that do not incorporate papillomavirus capsid protein L2 into capsomeres of papillomavirus capsid protein L1 is that requirements for L1 capsomers to assemble into VLPs are somewhat strict, i.e. acidic pH, such as pH 5.2, is required for assembly. Thus, if it is desired to produce VLPs, it is desirable to produce capsomeres that assemble more readily into VLPs, i.e. at more physiological pH.
Currently, one approach to developing a therapeutic vaccine for treating established cervical carcinomas has been to create VLPs using papillomavirus capsid protein L1 also containing an immunotherapeutic protein, typically E7. See Miller, et al., Virology, 234:93-111. However, this approach does not include papillomavirus capsid protein L2, which can broaden the therapeutic usefulness of such a vaccine. Another drawback to this approach is that smaller amounts of immunotherapeutic protein can be incorporated into VLPs using this approach, certainly much less than one therapeutic protein per capsomere.
Thus, there remains a need in the art to produce papillomavirus vaccines containing both L1 and L2 viral capsid proteins to potentially generate a broader spectrum of protection against different papillomavirus serotypes. Additionally, there remains a need in the art to produce a therapeutic vaccine in order to treat established cervical cancer which represents an improvement over current therapeutic vaccines. There remains a need for such compositions to be produced economically, preferably from bacterial expression systems. Further, there is a need in the art to for bacterially produced papillomavirus capsomeres that assemble into VLPs at physiologic pH.